This invention is in the area of the expression of catalytically active Memapsin 2 (beta-secretase) and use thereof in the design and screening of specific inhibitors which are useful in the treatment and/or prevention of Alzheimer""s Disease.
Alzheimer""s disease (AD) is a degenerative disorder of the brain first described by Alios Alzheimer in 1907 after examining one of his patients who suffered drastic reduction in cognitive abilities and had generalized dementia (The early story of Alzheimer""s Disease, edited by Bick et al. (Raven Press, N.Y. 1987)). It is the leading cause of dementia in elderly persons. AD patients have increased problems with memory loss and intellectual functions which progress to the point where they cannot function as normal individuals. With the loss of intellectual skills the patients exhibit personality changes, socially inappropriate actions and schizophrenia (A Guide to the Understanding of Alzheimer""s Disease and Related Disorders, edited by Jorm (New York University Press, New York 1987). AD is devastating for both victims and their families, for there is no effective palliative or preventive treatment for the inevitable neurodegeneration.
AD is associated with neuritic plaques measuring up to 200 xcexcm in diameter in the cortex, hippocampus, subiculum, hippocampal gyrus, and amygdala. One of the principal constituents of neuritic plaques is amyloid, which is stained by Congo Red (Fisher (1983); Kelly Microbiol. Sci. 1(9):214-219 (1984)). Amyloid plaques stained by Congo Red are extracellular, pink or rust-colored in bright field, and birefringent in polarized light. The plaques are composed of polypeptide fibrils and are often present around blood vessels, reducing blood supply to various neurons in the brain.
Various factors such as genetic predisposition, infectious agents, toxins, metals, and head trauma have all been suggested as possible mechanisms of AD neuropathy. Available evidence strongly indicates that there are distinct types of genetic predispositions for AD. First, molecular analysis has provided evidence for mutations in the amyloid precursor protein (APP) gene in certain AD-stricken families (Goate et al. Nature 349:704-706 (1991); Murrell et al. Science 254:97-99 (1991); Chartier-Harlin et al. Nature 353:844-846 (1991); Mullan et al., Nature Genet. 1:345-347 (1992)). Additional genes for dominant forms of early onset AD reside on chromosome 14 and chromosome 1 (Rogaev et al., Nature 376:775-778 (1995); Levy-Lahad et al., Science 269:973-977 (1995); Sherrington et al., Nature 375:754-760 (1995)). Another loci associated with AD resides on chromosome 19 and encodes a variant form of apolipoprotein E (Corder, Science 261:921-923 (1993)).
Amyloid plaques are abundantly present in AD patients and in Down""s Syndrome individuals surviving to the age of 40. The overexpression of APP in Down""s Syndrome is recognized as a possible cause of the development of AD in Down""s patients over thirty years of age (Rumble et al., New England J. Med. 320:1446-1452 (1989); Mann et al., Neurobiol. Aging 10:397-399 (1989)). The plaques are also present in the normal aging brain, although at a lower number. These plaques are made up primarily of the amyloid xcex2 peptide (Axcex2; sometimes also referred to in the literature as xcex2-amyloid peptide or xcex2 peptide) (Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890 (1984)), which is also the primary protein constituent in cerebrovascular amyloid deposits. The amyloid is a filamentous material that is arranged in beta-pleated sheets. Axcex2 is a hydrophobic peptide comprising up to 43 amino acids.
The determination of its amino acid sequence led to the cloning of the APP cDNA (Kang et al., Nature 325:733-735 (1987); Goldgaber et al., Science 235:877-880 (1987); Robakis et al., Proc. Natl. Acad. Sci. 84:4190-4194 (1987); Tanzi et al., Nature 331:528-530 (1988)) and genomic APP DNA (Lemaire et al., Nucl. Acids Res. 17:517-522 (1989); Yoshikai et al., Gene 87, 257-263 (1990)). A number of forms of APP cDNA have been identified, including the three most abundant forms, APP695, APP751, and APP770. These forms arise from a single precursor RNA by alternate splicing. The gene spans more than 175 kb with 18 exons (Yoshikai et al. (1990)). APP contains an extracellular domain, a transmembrane region and a cytoplasmic domain. Axcex2 consists of up to 28 amino acids just outside the hydrophobic transmembrane domain and up to 15 residues of this transmembrane domain. Axcex2 is normally found in brain and other tissues such as heart, kidney and spleen. However, Axcex2 deposits are usually found in abundance only in the brain.
Van Broeckhaven et al., Science 248:1120-1122 (1990), have demonstrated that the APP gene is tightly linked to hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) in two Dutch families. This was confirmed by the finding of a point mutation in the APP coding region in two Dutch patients (Levy et al., Science 248:1124-1128 (1990)). The mutation substituted a glutamine for glutamic acid at position 22 of the Axcex2 (position 618 of APP695, or position 693 of APP770). In addition, certain families are genetically predisposed to Alzheimer""s disease, a condition referred to as familial Alzheimer""s disease (FAD), through mutations resulting in an amino acid replacement at position 717 of the full length protein (Goate et al. (1991); Murrell et al. (1991); Chartier-Harlin et al. (1991)). These mutations co-segregate with the disease within the families and are absent in families with late-onset AD. This mutation at amino acid 717 increases the production of the Axcex21-42 form of Axcex2 from APP (Suzuki et al., Science 264:1336-1340 (1994)). Another mutant form contains a change in amino acids at positions 670 and 671 of the full length protein (Mullan et al. (1992)). This mutation to amino acids 670 and 671 increases the production of total Axcex2 from APP (Citron et al., Nature 360:622-674 (1992)).
APP is processed in vivo at three sites. The evidence suggests that cleavage at the xcex2-secretase site by a membrane associated metalloprotease is a physiological event. This site is located in APP 12 residues away from the lumenal surface of the plasma membrane. Cleavage of the xcex2-secretase site (28 residues from the plasma membrane""s lumenal surface) and the xcex2-secretase site (in the transmembrane region) results in the 40/42-residue xcex2-amyloid peptide (A xcex2), whose elevated production and accumulation in the brain are the central events in the pathogenesis of Alzheimer""s disease (for review, see Selkoe, D. J. Nature 399:23-31 (1999)). Presenilin 1, another membrane protein found in human brain, controls the hydrolysis at the APP (xcex2-secretase site and has been postulated to be itself the responsible protease (Wolfe, M. S. et al., Nature 398:513-517 (1999)). Presenilin 1 is expressed as a single chain molecule and its processing by a protease, presenilinase, is required to prevent it from rapid degradation (Thinakaran, G. et al., Neuron 17:181-190 (1996) and Podlisny, M. B., et al., Neurobiol. Dis. 3:325-37 (1997)). The identity of presenilinase is unknown. The in vivo processing of the xcex2-secretase site is thought to be the rate-limiting step in A xcex2 production (Sinha, S. and Lieberburg, I., Proc. Natl. Acad. Sci., USA, 96:11049-11053 (1999)), and is therefore a strong therapeutic target.
The design of inhibitors effective in decreasing amyeloid plaque formation is dependent on the identification of the critical enzyme(s) in the cleavage of APP to yield the 42 amino acid peptide, the Axcex21-42 form of Axcex2. Although several enzymes have been identified, it has not been possible to produce active enzyme. Without active enzyme, one cannot confirm the substrate specificity, determine the subsite specificity, nor determine the kinetics or critical active site residues, all of which are essential for the design of inhibitors.
Memapsin 2 has been shown to be beta-secretase, a key protease involved in the production in human brain of beta-amyloid peptide from beta-amyloid precursor protein (for review, see Selkoe, D. J. Nature 399:23-31 (1999)). It is now generally accepted that the accumulation of beta- amyloid peptide in human brain is a major cause for the Alzheimer""s disease. Inhibitors specifically designed for human memapsin 2 should inhibit or decrease the formation of beta-amyloid peptide and the progression of the Alzheimer""s disease.
Memapsin 2 belongs to the aspartic protease family. It is homologous in amino acid sequence to other eukaryotic aspartic proteases and contains motifs specific to that family. These structural similarities predict that memapsin 2 and other eukaryotic aspartic proteases share common catalytic mechanism Davies, D. R., Annu. Rev. Biophys. Chem. 19, 189 (1990). The most successful inhibitors for aspartic proteases are mimics of the transition state of these enzymes. These inhibitors have substrate-like structure with the cleaved planar peptide bond between the carbonyl carbon and the amide nitrogen replaced by two tetrahedral atoms, such as hydroxyethylene [xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94], which was originally discovered in the structure of pepstatin (Marciniszyn et al., 1976).
However, for clinical use, it is preferable to have small molecule inhibitors which will pass through the blood brain barrier and which can be readily synthesized. It is also desirable that the inhibitors are relatively inexpensive to manufacture and that they can be administered orally. Screening of thousands of compounds for these properties would require an enormous effort. To rationally design memapsin 2 inhibitors for treating Alzheimer""s disease, it will be important to know the three-dimensional structure of memapsin 2, especially the binding mode of an inhibitor in the active site of this protease.
It is therefore an object of the present invention to provide purified, recombinant, and active memapsin 2, as well as its substrate and subsite specificity and critical active site residues.
It is a further object of the present invention to provide compositions and methods for synthesis of inhibitors of memapsin 2.
It is a still further object of the present invention to provide compositions that interact with memapsin 2 or its substrate to inhibit cleavage by the memapsin 2 which can cross the blood brain barrier (BBB).
It is therefore an object of the present invention to provide means for rational design and screening of compounds for inhibition of mamapsin 2.
Methods for the production of purified, catalytically active, recombinant memapsin 2 have been developed. The substrate and subsite specificity of the catalytically active enzyme have been determined. The active enzyme and assays for catalytic activity are useful in screening libraries for inhibitors of the enzyme.
The substrate and subsite specificity information was used to design substrate analogs of the natural memapsin 2 substrate that can inhibit the function of memapsin 2. The substrate analogs are based on peptide sequences, shown to be related to the natural peptide substrates for memapsin 2. The substrate analogs contain at least one analog of an amide (peptide) bond which is not capable of being cleaved by memapsin 2. Processes for the synthesis of two substrate analogues including isosteres at the sites of the critical amino acid residues were developed and the substrate analogues, OMR99-1 and OM99-2, were synthesized. OM99-2 is based on an octapeptide Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe (SEQ ID NO:28) with the Leu-Ala peptide bond substituted by a transition-state isostere hydroxyethylene group. The inhibition constant of OM99-2 is 1.6xc3x9710xe2x88x929M against recombinant pro-memapsin 2. Crystallography of memapsin 2 bound to this inhibitor was used to determine the three dimensional structure of the protein, as well as the importance of the various residues in binding.
This information can be used by those skilled in the art to design new inhibitors, using commercially available software programs and techniques familiar to those in organic chemistry and enzymology, to design new inhibitors. For example, the side chains of the inhibitors may be modified to produce stronger interactions (through hydrogen bonding, hydrophobic interaction, charge interaction and/or van der Waal interaction) in order to increase inhibition potency. Based on this type of information, the residues with minor interactions may be eliminated from the new inhibitor design to decrease the molecular weight of the inhibitor. The side chains with no structural hindrance from the enzyme may be cross-linked to lock in the effective inhibitor conformation. This type of structure also enables the design of peptide surrogates which may effectively fill the binding sites of memapsin 2 yet produce better pharmaceutical properties.
The examples demonstrate the production of catalytically active enzyme, design and synthesis of inhibitors, and how the crystal structure was obtained. The examples thereby demonstrate how the methods and materials described herein can be used to screen libraries of compounds for other inhibitors, as well as for design of inhibitors. These inhibitors are useful in the prevention and/or treatment of Alzheimer""s disease as mediated by the action of the beta secretase memapsin 2, in cleaving APP.